Foamy viruses comprise a subfamily of retroviruses with many unique features. They are the most ancient vertebrate RNA viruses and are ubiquitous in non-human primates. They are also known to infect small numbers of humans. Aspects of their replication pathway are quite similar to that of the hepadnaviruses, for example, conversion of RNA to a DNA genome during assembly and requirement for only a single capsid protein. However, unlike members of both groups which can cause cancers and immunodeficiencies, foamy viruses are non-pathogenic. This feature makes them attractive candidates for gene therapy vectors, and development of foamy viral vectors is underway. Such foamy virus vectors show promise for cancer, infectious diseases and genetic disorders because of their broad host range and ability to infect early stem cells. However, there are many gaps in our knowledge of how these viruses replicate. The goal of this work is to increase understanding of foamy virus assembly and gene regulation. We will focus on 3 aspects;viral capsid assembly and association with viral glycoproteins, packaging of the viral genome and enzymes, and regulation of splicing of the viral Pol mRNA. At each of these replication steps, foamy viruses are different from both orthoretroviruses and hepadnaviruses. We will take both molecular genetic and cell biology approaches to study viral replication, including analysis of viral mutants, yeast 2-hybrid assays, immunofluorescence and immunoelectron microscopy. In Aim 1, we will analyze the cellular sites for viral assembly, the host and viral protein interactions that are required, and the regions of the structural protein (Gag) that interact with the viral glycoproteins to allow viral budding. In Aim 2, we will determine the regions of Gag that are important for encapsidation of the viral genome and the viral Pol protein (that encodes all of the viral enzymes). In Aim 3, we will examine how splicing of the unique mRNA encoding Pol is regulated. Completion of these Aims will help in the design of foamy virus vectors and increase our knowledge about the diverse pathways possible for retroviral replication.